Isolated human dehydrogenases, nucleic acid molecules encoding these human dehydrogenases, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of polypeptides that are encoded by genes within the human genome, the dehydrogenase polypeptides of the present invention. The present invention specifically provides isolated polypeptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the dehydrogenase polypeptides, and methods of identifying modulators of the dehydrogenase polypeptides.

FIELD OF THE INVENTION

The present invention is in the field of dehydrogenases that are relatedto the retinol dehydrogenase subfamily, recombinant DNA molecules andprotein production. The present invention specifically provides noveldehydrogenase polypeptides and proteins and nucleic acid moleculesencoding such peptide and protein molecules, all of which are useful inthe development of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

Dehydrogenases, particularly members of the retinol dehydrogenasesubfamilies, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thesesubfamily of dehydrogenases. The present invention advances the state ofthe art by providing a previously unidentified human dehydrogenases thathave homology to members of the retinol dehydrogenase subfamilies.

DEHYDROGENASES

17.beta.-hydroxysteroid Dehydrogenase

The enzymes identified as 17.beta.-hydroxysteroid dehydrogenase (HSD)are important in the production of human sex steroids, includingandrost-5-ene-3.beta.,17.beta.-diol (.DELTA.sup.5-diol), testosteroneand estradiol. In humans, several types of 17.beta.-HSD have now beenidentified and characterized. Each type of 17.beta.-HSD has been foundto catalyze specific reactions and to be located in specific tissues.Further information about Types 1, 2 and 3 17.beta.-HSD can be had byreference as follows: Type 1 17.beta.-HSD is described by Luu-The, V. etal., Mol. Endocrinol., 3:1301-1309 (1989) and by Peltoketo, H. et al.,FEBSLett, 239:73-77 (1988); Type 2 17.beta.-HSD is described in Wu, L.et al., J. Biol Chem, 268:12964-12969 (1993); Type 3 17.beta.-HSD isdescribed in Geissler, W. M., Nature Genetics, 7:34-39 (1994).

Ihibitors of 17.beta.-hydroxysteroid dehydrogenase activity can be usedfor prophylaxis or treatment of benign prostate hypertrophy (see WOpublication 91/100731).

20-alpha-hydroxysteroid Dehydrogenase

The enzyme responsible for the ovarian metabolism of progesterone to20.alpha.-hydroxyprogesterone is 20.alpha.-hydroxysteroid dehydrogenase(20.alpha.-HSD). Specifically, 20.alpha.-HSD is nicotinamide adeninedinucleotide phosphate (NADPH)-dependent and catalyzes the transfer ofhydrogen from NADPH to progesterone.

By metabolizing progesterone to an inactive form, 20.alpha.-HSD plays acentral role in inhibiting the maintenance of pregnancy and preventionof implantation [Wiest, Endocrinology 83:1181-184 (1968); Wiest et al.,Endocrinology 82:844-859 (1968); Kuhn and Briley, i Biochem. J.0117:193-200 (1970); Rodway and Kuhn, Biochem. J. 152:433-443 (1975)].Further supporting this role is the fact that it is the increase inovarian 20.alpha.-HSD activity rather than a decrease in the synthesisof progesterone that contributes to the lower circulating progesteronelevels associated with the termination of pregnancy [Kuhn and Briley,Biochem. J. 117:193-201 (1970)]. Indeed, 20.alpha.-HSD gene expression[Albarracin et al. Endocrinology 134:2453-2460 (1994)] and activityremains repressed throughout pregnancy but are induced beforeparturition [Wiest et al., Endocrinology 82:844-859 (1968); Kuhn andBriley, Biochem. J. 117:193-200 (1970]. Also, ovarian 20.alpha.-HSDcatalyzes the decline in progesterone levels which occur during normaland induced termination of pregnancy and pseudopregnancy [Hashimoto andWiest, Endocrinology 84:873-885 (1969); Naito et al., Endocrinology Jpn33(1):43-50 (February 1986)].

While 20.alpha.-HSD is of much interest as a key enzyme in thetermination/prevention of pregnancy, it is possible that the enzyme isalso of importance in spontaneous abortions. Specifically, it ispossible that a significant number of spontaneous abortions are due toearly expression of 20.alpha.-HSD. Therefore, detection of early20.alpha.-HSD expression would be of interest in those susceptible toearly spontaneous abortions. If detection is made early enough,progesterone replacement therapy could be initiated to help maintain thepregnancy.

11.beta.-hydroxysteroid Dehydrogenase

Corticosteroids, also referred to as glucocorticoids, are steroidhormones, the most common form of which is cortisol. Modulation ofglucocorticoid activity is important in regulating physiologicalprocesses in a wide range of tissues and organs. Glucocorticoids actwithin the gonads to directly suppress testosterone production (Monderet al., 1994). High levels of glucocorticoids may also result inexcessive salt and water retention by the kidneys, producing high bloodpressure.

Glucocorticoid action is mediated via binding of the molecule to areceptor, such as either a mineralocorticoid receptor (MR) or aglucocorticoid receptor (GR). Krozowski et al. (1983) and Beaumont andFanestil (1983) showed that MR of adrenalectomised rats have an equalaffinity for the mineralocorticoid aldosterone and glucocorticoids, forexample corticosterone and cortisol. Confirmatory evidence has beenfound for human MR (Arriza et al., 1988). In patients suffering from thecongenital syndrome of Apparent Mineralocorticoid Excess (AME; Ulick etal., 1979), cortisol levels are elevated and bind to and activate MRsnormally occupied by aldosterone, the steroid that regulates salt andwater balance in the body. Salt and water are retained in AME patientscausing severe hypertension.

The enzyme 11.beta.-hydroxysteroid dehydrogenase (11.beta.HSD) convertsglucocorticoids into metabolites that are unable to bind to MRs (Edwardset al., 1988; Funder et al., 1988), present in mineralocorticoid targettissues, for example kidney, pancreas, small intestine, colon, as wellas the hippocampus, placenta and gonads. For example, in aldosteronetarget tissues 11.beta.HSD inactivates glucocorticoid molecules,allowing the much lower circulating levels of aldosterone to maintainrenal homeostasis. When the 11.beta.HSD enzyme is inactivated, forexample in AME patients (Ulick et al., 1979) or following administrationof glycyrrhetinic acid, a component of licorice, severe hypertensionresults. Further, placental 11.beta.HSD activity may protect the foetusfrom high circulating levels of glucocorticoid which may predispose tohypertension in later life (Edwards et al., 1993).

Biochemical characterisation of 11.beta.HSD activity indicates thepresence of at least two isoenzymes (11.beta.HSD1 and 11.beta.HSD2) withdifferent cofactor requirements and substrate affnities. The 1.betaHSD1and 11.beta.HSD2) with differnt cofactor that prefers NADP+ as acofactor (Agarwal et al., 1989). The 11.beta.HSD2 enzyme is a highaffinity enzyme (Km for glucocorticoid=10 nM), requiring NAD+, not NADP+as the preferred cofactor, belonging to a class of glucocorticoiddehydrogenase enzymes hereinafter referred to as “NAD+ dependentglucocorticoid dehydrogenase” enzymes.

Michael et al. (1993) show an inverse correlation between 11.beta.HSDenzyme activity in human granulosa-lutein cells and the success of IVF(in vitro fertilization), and suggest that activity of this enzyme mightbe related to the success of embryo attachment and implantationfollowing IVF. The measurement of ovarian 11.beta.HSD enzyme activity asa prognostic indicator for the outcome of assisted conception in allspecies, is the subject of UK Patent Application No 9305984.

3alpha-hydroxysteroid Dehydrogenase

Human liver 3alpha-hydroxysteroid plays an important role in themetabolism of steroid hormones and polycyclic aromatic hydrocarbons andin the reduction of ketone-containing drugs (Kume et al.,Pharmacogenetics December 1999; 9(6):763-71). 3alpha-hydroxysteroid isalso involved in the metabolism of bile acids (Yamamoto et al., BiolPharm Bull November 1998; 21(11):1148-53).

3alpha-hydroxysteroid plays a significant role in5alpha-dihydrotestosterone metabolism in human liver via3alpha-hydroxysteroid reduction, followed by subsequent glucuronidationand clearance via the kidney (Pirog et al., J Clin Endocrinol MetabSeptember 1999; 84(9):3217-21).

Trans-1,2-dihydrobenzene-1,2-diol Dehydrogenase

Two major forms of trans-1,2-dihydrobenzene-1,2-diol dehydrogenaseexist. One form shows strict specificity for benzene dihydrodiol andNADP+. The other form oxidizes n-butanol, glycerol, sorbitol, andbenzene dihydrodiol in the presence of NADP+ or NAD+, and exhibits highreductase activity towards aldehydes, aldoses and diacetyls (Matsuura etal., Biochim Biophys Acta April 1987 8;912(2):270-7).

3-oxo-5-beta-steroid 4 dehydrogenase (also referred to as delta4-3-Ketosteroid 5 beta-reductase)

3-oxo-5-beta-steroid 4 dehydrogenase exhibits activity toward a varietyof substrates, including testosterone, cortisol, cortisone,progesterone, 4-androstene-3,17-dione, 7alpha-hydroxy-4-cholesten-3-one, and 7 alpha, 12alpha-dihydroxy-4-cholesten-3-one (Okuda et al., J Biol Chem June 198425;259(12):7519-24).

Retinol Dehydrogenase

Vitamin A is a pigment essential to vision. Vitamin A comes from theenzymatic conversion of carotenoids, yellow pigments common to carrotsand other vegetables, to retinol. Deficiency of vitamin A andinsufficient retinol production leads to a variety of maladies in humansand experimental animals. Symptoms of deficiency include vision relateddisorders such as xerophthalmia and night blindness; dry skin and drymucous membranes; retarded development and growth; and sterility in maleanimals.

Cleavage of .beta.-carotene yields two molecules of retinol; oxidationof retinol forms retinal. Retinal and opsin combine to producerhodopsin, a visual pigment found in nature. The excitation of rhodopsinwith visible light triggers a series of photochemical reactions andconfornational changes in the molecule which result in the electricalsignal to the brain that are the basis of visual transduction (Lehningeret al. (1993) Principles of Biochemistry, Worth Publishers, New York,N.Y.).

Retinol dehydrogenase (RoDH) catalyzes the conversion of retinol toretinal; retinal dehydrogenase converts retinal to retinoate. Retinoateis a retinoid and a hormone which controls numerous biological processesby regulating eukaryotic gene expression. Retinoids, like steroid andthyroid hormones, diffuse directly across the plasma membrane and bindto intracellular receptor proteins. Binding activates the receptorswhich interact with signaling pathways (Vettermann et al. (1997) Mol.Carcinog. 20: 58-67), and regulate the transcription of specific genes,particularly those mediating vertebrate development (Alberts et al.(1994) Molecular Biology of the Cell, Garland Publishing, Inc., NewYork, N.Y.). Retinol is known to be important in epithelial development(Haselbeck et al. (1997) Dev. Dyn. 208: 447-453; and Attar et al. (1997)Mol. Endocrinol. 11: 792-800) and in the development of the centralnervous system (Maden et al. (1997) Development 124: 2799-2805). InMaden's studies on quail embryos, absence of vitamin A, lead to severedeficits including lack of a posterior hindbrain. Conversely, injectionof retinol before gastrulation of the embryo prevented positionalapoptosis and corrected the CNS defects.

The universal chromophore of visual pigments is 11-cis retinaldehydewhich is generated by 11-cis retinol dehydrogenase, a membrane-boundenzyme abundantly expressed in the retinal pigment epithelium of theeye. The gene which encodes 11-cis retinol dehydrogenase may be involvedin hereditary eye diseases (Simon et al. (1996) Genomics 36: 424-430).

Chai et al. have identified, cloned, and expressed two isoforms ofretinol dehydrogenase, RoDH(l) and (RoDH(II) (1995, J. Biol. Chem. 270:28408-28412). The deduced amino acid sequence shows that RoDH(I) andRoDH(II) are short-chain dehydrogenases/reductases that share 82%identity. Retinol is the substrate for RoDH(II) which has a higheraffinity for NADP than NAD and is stimulated by ethanol and phosphatidylcholine. Although RoDH(II) is not inhibited by the medium-chain alcoholdehydrogenase inhibitor, 4-methylpyrazole, it is inhibited byphenylarsine oxide and carbenoxolone. Chai et al. reported detection ofRoDH(I) and RoDH(II) mRNA in rat liver, but RNase protection assaysrevealed RoDH(I) and RoHD(II) MRNA in kidney, lung, testis, and brain.Based on these data, Chai et al. concluded that RoDH has tissue specificexpression.

The retinol signaling pathway plays an important role in human disordersand diseases. Retinoic acid receptors (RARs; -alpha, -beta, and -gamma)are retinoid-activated transcription factors, which mediate effects ofretinoids on gene expression. Alterations in receptor expression orfunction could interfere with the retinoid signaling pathway.Interference with the pathway may enhance cancer development. Vitamin Aanalogs (retinoids) which interact with RARs, suppress oral and lungcarcinogenesis in animal models and prevent the development of tumors inhead, neck, and lung cancer patients (Lotan R. 1997 Environ. HeathPerspect. 105 Suppl. 4: 985-988). Lotan reported that RAR betaexpression is lost at early stages of carcinogenesis in theaerodigestive tract.

Retinol dehydrogenase may be implicated in embryonic development. Thestudies of Maden et al. (supra) suggest that retinol may play asignificant role in controlling apoptosis during development of thecentral nervous system. Retinoids are also implicated in epidermaldevelopment. Attar et al. (1997, Mol. Endocrinol. 11: 792-800) showedthat disruption of epidermal barrier function results in extremely highincidences of neonatal mortality in pups.

In addition, retinol dehydrogenase activity is linked to hereditary eyediseases (Simon et al. (1996) Genomics 36: 424-430). Autosomal recessivechildhood-onset severe retinal dystrophy (arCSRD) is a heterogeneousgroup of disorders that affect rod and cone photoreceptorssimultaneously. Disease genes implicated in arCSRD are expected toencode proteins present in the neuroretina or in the retinal pigmentepithelium (RPE). RPE65, a tissue-specific and evolutionarily highlyconserved 61 kD protein, is the first disease gene in this group ofinherited disorders that is expressed exclusively in RPE, and may play arole in vitamin A metabolism of the retina (Gu et al. (1997) Nat. Genet.17: 194-197).

Pityriasis rubra pilaris (PRP) is an idiopathic erythematous scalingeruption which can be difficult to distinguish from psoriasis. Theexpression of RoDH(II) in the retinol signaling pathway may be ofpathogenetic importance in the diagnosis of PRP (Magro, C. M. andCrowson, A. N. (1997) J. Cutan. Pathol. 24: 416-424).

The discovery of a new human retinol dehydrogenase and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention and treatmentof disorders associated with immune response, cell proliferation, anddevelopment.

Substantial chemical and structural homology exists between the proteindescribed herein and 11-cis retinol dehydrogenase (see FIG. 1). 11-cisretinol dehydrogenase are known in the art to be involved in retinaldegeneration. For more information relating to the protein of thepresent invention, see Simon et al., Genomics September 199615;36(3):424-30A, Yamamoto et al., Nat Genet June 1999; 22(2):188-91H.

Dehydrogenase proteins, particularly members of the retinoldehydrogenase subfamily, are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown members ofthis subfamily of dehydrogenase proteins. The present invention advancesthe state of the art by providing a previously unidentified humandehydrogenase proteins that have homology to members of the retinoldehydrogenase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human dehydrogenase polypeptides and proteins that arerelated to the 11-cis retinol dehydrogenase, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate dehydrogenaseactivity in cells and tissues that express the dehydrogenase.Experimental data as provided in FIG. 1 indicates expression in themalignant melanoma (metastatic to lymph node), brain (glioblastoma),thyroid, colon tumor (RER+), stomach (poorly differentiatedadenocarcinoma with signet ring cell features), primary B-cells fromtonsils, lung carcinoid, Burkitt lymphoma and human leukocyte.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule sequence thatencodes the dehydrogenase of the present invention. (SEQ ID NO:1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte.

FIG. 2 provides the predicted amino acid sequence of the dehydrogenaseof the present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thedehydrogenase of the present invention. (SEQ ID NO:3) In additionstructure and fnuctional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 11 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a dehydrogenase or part of a dehydrogenase and arerelated to the retinol dehydrogenase subfamily. Utilizing thesesequences, additional genonic sequences were assembled and transcriptand/or cDNA sequences were isolated and characterized. Based on thisanalysis, the present invention provides amino acid sequences of humandehydrogenase polypeptides that are related to the retinol dehydrogenasesubfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these dehydrogenasepolypeptide, nucleic acid variation (allelic information), tissuedistribution of expression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thedehydrogenase of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known dehydrogenases of the retinoldehydrogenase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in themalignant melanoma (metastatic to lymph node), brain (glioblastoma),thyroid, colon tumor (RER+), stomach (poorly differentiatedadenocarcinoma with signet ring cell features), primary B-cells fromtonsils, lung carcinoid, Burkitt lymphoma and human leukocyte. The arthas clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known retinol dehydrogenase family or subfamily ofdehydrogenases.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thedehydrogenase family and are related to the retinol dehydrogenasesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the dehydrogenases or peptides of the present invention,dehydrogenases or peptides, or peptides/proteins of the presentinvention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the dehydrogenase polypeptide disclosed in the FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNAor FIG. 3, genomic sequence), as well as all obvious variants of thesepeptides that are within the art to make and use. Some of these variantsare described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components.

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thedehydrogenase polypeptide having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

The isolated dehydrogenase polypeptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inthe malignant melanoma (metastatic to lymph node), brain (glioblastoma),thyroid, colon tumor (RER+), stomach (poorly differentiatedadenocarcinoma with signet ring cell features), primary B-cells fromtonsils, lung carcinoid, Burkitt lymphoma and human leukocyte. Forexample, a nucleic acid molecule encoding the dehydrogenase polypeptideis cloned into an expression vector, the expression vector introducedinto a host cell and the protein expressed in the host cell. The proteincan then be isolated from the cells by an appropriate purificationscheme using standard protein purification techniques. Many of thesetechniques are described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genornic sequences provided inFIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the dehydrogenase polypeptide of the present invention arethe naturally occurring mature proteins. A brief description of howvarious types of these proteins can be made/isolated is provided below.

The dehydrogenase polypeptides of the present invention can be attachedto heterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a dehydrogenase polypeptideoperatively linked to a heterologous protein having an amino acidsequence not substantially homologous to the dehydrogenase polypeptide.“Operatively linked” indicates that the dehydrogenase polypeptide andthe heterologous protein are fused in-frame. The heterologous proteincan be fused to the N-terminus or C-terminus of the dehydrogenasepolypeptide.

In some uses, the fusion protein does not affect the activity of thedehydrogenase polypeptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant dehydrogenase polypeptide. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a protein can beincreased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A dehydrogenase polypeptide-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the dehydrogenase polypeptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the peptides of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art know techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the dehydrogenase polypeptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily, and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, word length=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the proteins ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See htto://www.ncbi.nlm.nih.gov.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thedehydrogenase polypeptides of the present invention as well as beingencoded by the same genetic locus as the dehydrogenase polypeptideprovided herein.

Allelic variants of a dehydrogenase polypeptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the dehydrogenasepolypeptide as well as being encoded by the same genetic locus as thedehydrogenase polypeptide provided herein. Genetic locus can readily bedetermined based on the genomic information provided in FIG. 3, such asthe genomic sequence mapped to the reference human. As used herein, twoproteins (or a region of the proteins) have significant homology whenthe amino acid sequences are typically at least about 70-80%, 80-90%,and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a dehydrogenase polypeptide encoding nucleic acid moleculeunder stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the transporter protein of the present invention. SNPs wereidentified at 11 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements. The changes in the amino acidsequence that these SNPs cause can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as abase.

Paralogs of a dehydrogenase polypeptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the dehydrogenase polypeptide, as being encoded by a genefrom humans, and as having similar activity or function. Two proteinswill typically be considered paralogs when the amino acid sequences aretypically at least about 40-50%, 50-60%, and more typically at leastabout 60-70% or more homologous through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a dehydrogenase polypeptide encoding nucleic acid molecule undermoderate to stringent conditions as more fully described below.

Orthologs of a dehydrogenase polypeptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the dehydrogenase polypeptide as well as being encoded by agene from another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a dehydrogenase polypeptide encodingnucleic acid molecule under moderate to stringent conditions, as morefully described below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the dehydrogenase polypeptides ofthe present invention can readily be generated using recombinanttechniques. Such variants include, but are not limited to deletions,additions and substitutions in the amino acid sequence of thedehydrogenase polypeptide. For example, one class of substitutions isconserved amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a dehydrogenase polypeptide by anotheramino acid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr, exchange of the acidic residues Asp and Glu, substitutionbetween the amide residues Asn and Gln, exchange of the basic residuesLys and Arg, replacements among the aromatic residues Phe, Tyr, and thelike. Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Variant dehydrogenase polypeptides can be fully functional or can lackfunction in one or more activities. Fully functional variants typicallycontain only conservative variations or variations in non-criticalresidues or in non-critical regions. Functional variants can alsocontain substitution of similar amino acids that result in no change oran insignificant change in function. Alternatively, such substitutionsmay positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.Sites that are critical for ligand-receptor binding can also bedetermined by structural analysis such as crystallography, nuclearmagnetic resonance, or photoalfinity labeling (Smith et al, J. Mol.Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the dehydrogenasepolypeptides, in addition to proteins and peptides that comprise andconsist of such fragments. Particularly those comprising the residuesidentified in FIG. 2. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that havebeen disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16 or morecontiguous amino acid residues from a dehydrogenase polypeptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the dehydrogenase polypeptide, or can bechosen for the ability to perform a function, e.g., act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example about 8 or more amino acids in lengthSuch fragments will typically comprise a domain or motif of thedehydrogenase polypeptide, e.g., active site. Further, possiblefragments include, but are not limited to, domain or motif containingfragments, soluble peptide fragments, and fragments containingimmunogenic structures. Predicted domains and functional sites arereadily identifiable by computer programs well known and readilyavailable to those of skill in the art (e.g., PROSITE, HMMer, eMOTIF,etc.). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in dehydrogenasepolypeptides are described in basic texts, detailed monographs, and theresearch literature, and they are well known to those of skill in theart (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross4inking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachmentsulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the dehydrogenase polypeptides of the present inventionalso encompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature dehydrogenase polypeptide isfused with another compound, such as a compound to increase thehalf-life of the dehydrogenase polypeptide (for example, polyethyleneglycol), or in which the additional amino acids are fused to the maturedehydrogenase polypeptide, such as a leader or secretory sequence or asequence for purification of the mature dehydrogenase polypeptide, or apro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in assays to determinethe biological activity of the protein, including in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother immune response; as a reagent (including the labeled reagent) inassays designed to quantitatively determine levels of the protein (orits ligand or receptor) in biological fluids; and as markers for tissuesin which the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state). Where the protein binds orpotentially binds to another protein (such as, for example, in areceptor-ligand interaction), the protein can be used to identify thebinding partner so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these research utilities are capableof being developed into reagent grade or kit format forcommercialization as research products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, dehydrogenases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the dehydrogenase. Experimental data asprovided in FIG. 1 indicates that dehydrogenases of the presentinvention are expressed in the malignant melanoma (metastatic to lymphnode), brain (glioblastoma), thyroid, colon tumor (RER+), stomach(poorly differentiated adenocarcinoma with signet ring cell features),primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma detectedby a virtual northern blot. In addition, PCR-based tissue screeningpanel indicates expression in human leukocyte. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofdehydrogenases, particularly members of the retinol dehydrogenasesubfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Experimental data as provided in FIG. 1 indicatesexpression in the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. Such uses can readily be determined using the informationprovided herein, that which is known in the art, and routineexperimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to dehydrogenases that are related tomembers of the retinol dehydrogenase subfamily. Such assays involve anyof the known dehydrogenase functions or activities or properties usefulfor diagnosis and treatment of dehydrogenase-related conditions that arespecific for the subfamily of dehydrogenases that the one of the presentinvention belongs to, particularly in cells and tissues that express thedehydrogenase. Experimental data as provided in FIG. 1 indicates thatdehydrogenases of the present invention are expressed in the malignantmelanoma (metastatic to lymph node), brain (glioblastoma), thyroid,colon tumor (RER+), stomach (poorly differentiated adenocarcinoma withsignet ring cell features), primary B-cells from tonsils, lungcarcinoid, Burkitt lymphoma detected by a virtual northern blot. Inaddition, PCR-based tissue screening panel indicates expression in humanleukocyte.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the dehydrogenase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in the malignant melanoma (metastatic to lymphnode), brain (glioblastoma), thyroid, colon tumor (RER+), stomach(poorly differentiated adenocarcinoma with signet ring cell features),primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the dehydrogenase.

The polypeptides can be used to identify compounds that modulatedehydrogenase activity. Both the dehydrogenase of the present inventionand appropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thedehydrogenase. These compounds can be further screened against afunctional dehydrogenase to determine the effect of the compound on thedehydrogenase activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thedehydrogenase to a desired degree.

Therefore, in one embodiment, retinol dehydrogenase or a fragment orderivative thereof may be administered to a subject to prevent or treata disorder associated with an increase in apoptosis. Such disordersinclude, but are not limited to, AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis.

In another embodiment, a pharmaceutical composition comprising retinoldehydrogenase may be administered to a subject to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In still another embodiment, an agonist which is specific for retinoldehydrogenase may be administered to prevent or treat a disorderassociated with increased apoptosis including, but not limited to, thoselisted above.

In a further embodiment, a vector capable of expressing retinoldehydrogenase, or a fragment or a derivative thereof, may be used toprevent or treat a disorder associated with increased apoptosisincluding, but not limited to, those listed above.

In cancer, where retinol dehydrogenase promotes cell proliferation, itis desirable to decrease its activity. Therefore, in one embodiment, anantagonist of retinol dehydrogenase may be administered to a subject toprevent or treat cancer including, but not limited to, adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma,and, in particular, cancers of the adrenal gland, bladder, bone, bonemarrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus. In one aspect, an antibody specific for retinoldehydrogenase may be used directly as an antagonist, or indirectly as atargeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express retinol dehydrogenase.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding retinol dehydrogenase may be administered to asubject to prevent or treat a cancer including, but not limited to, thetypes of cancer listed above.

In inflammation, where retinol dehydrogenase promotes cellproliferation, it is desirable to decrease its activity. Therefore, inone embodiment, an antagonist of retinol dehydrogenase may beadministered to a subject to prevent or treat an inflammation. Disordersassociated with inflammation include, but are not limited to, Addison'sdisease, adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma In one aspect, anantibody specific for retinol dehydrogenase may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express retinoldehydrogenase.

Further, the dehydrogenase polypeptides can be used to screen a compoundfor the ability to stimulate or inhibit interaction between thedehydrogenase and a molecule that normally interacts with thedehydrogenase, e.g. a ligand or a component of the signal pathway thatthe dehydrogenase normally interacts. Such assays typically include thesteps of combining the dehydrogenase with a candidate compound underconditions that allow the dehydrogenase, or fragment, to interact withthe target molecule, and to detect the formation of a complex betweenthe protein and the target or to detect the biochemical consequence ofthe interaction with the dehydrogenase and the target, such as any ofthe associated effects of signal transduction.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries). (Hodgson, Bio/technology, September 1992,10(9);973-80).

One candidate compound is a soluble fragment of the dehydrogenase thatcompetes for ligand binding. Other candidate compounds include mutantdehydrogenases or appropriate fragments containing mutations that affectdehydrogenase function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, is withinthe scope of the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) dehydrogenase activity.The assays typically involve an assay of events in the dehydrogenasemediated signal transduction pathway that indicate dehydrogenaseactivity. Thus, the phosphorylation of a protein/ligand target, theexpression of genes that are up- or down-regulated in response to thedehydrogenase dependent signal cascade can be assayed. In oneembodiment, the regulatory region of such genes can be operably linkedto a marker that is easily detectable, such as luciferase.Alternatively, phosphorylation of the dehydrogenase, or a dehydrogenasetarget, could also be measured.

Any of the biological or biochemical functions mediated by thedehydrogenase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art.

Binding and/or activating compounds can also be screened by usingchimeric dehydrogenases in which any of the protein's domains, or partsthereof, can be replaced by heterologous domains or subregions.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which thedehydrogenase is derived.

The dehydrogenase polypeptide of the present invention is also useful incompetition binding assays in methods designed to discover compoundsthat interact with the dehydrogenase. Thus, a compound is exposed to adehydrogenase polypeptide under conditions that allow the compound tobind or to otherwise interact with the polypeptide. Solubledehydrogenase polypeptide is also added to the mixture. If the testcompound interacts with the soluble dehydrogenase polypeptide, itdecreases the amount of complex formed or activity from thedehydrogenase target. This type of assay is particularly useful in casesin which compounds are sought that interact with specific regions of thedehydrogenase. Thus, the soluble polypeptide that competes with thetarget dehydrogenase region is designed to contain peptide sequencescorresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the dehydrogenase, or fragment, or its target moleculeto facilitate separation of complexes from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase/15625 fusion proteins can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofdehydrogenase-binding protein found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques. For example,either the polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin with techniques wellknown in the art. Alternatively, antibodies reactive with the proteinbut which do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and the proteintrapped in the wells by antibody conjugation. Preparations of adehydrogenase-binding protein and a candidate compound are incubated inthe dehydrogenase-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thedehydrogenase target molecule, or which are reactive with dehydrogenaseand compete with the target molecule, as well as enzyme-inked assayswhich rely on detecting an enzymatic activity associated with the targetmolecule.

Agents that modulate one of the dehydrogenases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal/insect model system.Such model systems are well known in the art and can readily be employedin this context.

Modulators of dehydrogenase activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the dehydrogenase associated pathway, by treating cells that expressthe dehydrogenase. Experimental data as provided in FIG. 1 indicatesexpression in the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. These methods of treatment include the steps of administeringthe modulators of protein activity in a pharmaceutical composition asdescribed herein, to a subject in need of such treatment.

In yet another aspect of the invention, the dehydrogenases can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 (1993);Madura et al., J. Biol. Chem. 268:12046-12054 (1993); Bartel et al.,Biotechniques 14:920-924 (1993); Iwabuchi et al., Oncogene 8:1693-1696(1993); and Brent WO94/10300), to identify other proteins that bind toor interact with the dehydrogenase and are involved in dehydrogenaseactivity. Such dehydrogenase-binding proteins are also likely to beinvolved in the propagation of signals by the dehydrogenases ordehydrogenase targets as, for example, downstream elements of adehydrogenase-mediated signaling pathway, e.g., a pain signalingpathway. Alternatively, such dehydrogenase-binding proteins are likelyto be dehydrogenase inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a dehydrogenase isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a dehydrogenase-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., LacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the dehydrogenase.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a dehydrogenase modulating agent, an antisensedehydrogenase nucleic acid molecule, a dehydrogenase-specific antibody,or a dehydrogenase-binding partner) can be used in an animal or insectmodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent Alternatively, an agent identified as describedherein can be used in an animal or insect model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The dehydrogenases of the present invention are also useful to provide atarget for diagnosing a disease or predisposition to a disease mediatedby the peptide, Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in the malignant melanoma (metastatic to lymphnode), brain (glioblastoma), thyroid, colon tumor (RER+), stomach(poorly differentiated adenocarcinoma with signet ring cell features),primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. The method involves contacting a biological sample with acompound capable of interacting with the receptor protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells, and fluids present within a subject.

The peptides also are useful to provide a target for diagnosing adisease or predisposition to a disease mediated by the peptide,Accordingly, the invention provides methods for detecting the presence,or levels of, the protein in a cell, tissue, or organism. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the receptor protein such that the interaction can bedetected.

The peptides of the present invention also provide targets fordiagnosing active disease, or predisposition to a disease, in a patienthaving a variant peptide. Thus, the peptide can be isolated from abiological sample and assayed for the presence of a genetic mutationthat results in translation of an aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered receptor activity in cell-basedor cell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andinununofluorescence using a detection reagents, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques. Particularly useful are methods that detectthe allelic variant of a peptide expressed in a subject and methodswhich detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11) : 983-985 (1996)), and Linder, M. W.(Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of thesevariations result in severe toxicity of therapeutic drugs in certainindividuals or therapeutic failure of drugs in certain individuals as aresult of individual variation in metabolism. Thus, the genotype of theindividual can determine the way a therapeutic compound acts on the bodyor the way the body metabolizes the compound. Further, the activity ofdrug metabolizing enzymes effects both the intensity and duration ofdrug action. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the receptor protein in which one ormore of the receptor functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other ligand-binding regions that are moreor less active in ligand binding, and receptor activation. Accordingly,ligand dosage would necessarily be modified to maximize the therapeuticeffect within a given population containing a polymorphism. As analternative to genotyping, specific polymorphic peptides could beidentified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in themalignant melanoma (metastatic to lymph node), brain (glioblastoma),thyroid, colon tumor (RER+), stomach (poorly differentiatedadenocarcinoma with signet ring cell features), primary B-cells fromtonsils, lung carcinoid, Burkitt lymphoma and human leukocyte.Accordingly, methods for treatment include the use of the dehydrogenaseor fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe dehydrogenases. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or receptor/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoeythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment Experimental data as provided in FIG. 1 indicates thatdehydrogenases of the present invention are expressed in the malignantmelanoma (metastatic to lymph node), brain (glioblastoma), thyroid,colon tumor (RER+), stomach (poorly differentiated adenocarcinoma withsignet ring cell features), primary B-cells from tonsils, lungcarcinoid, Burkitt lymphoma detected by a virtual northern blot. Inaddition, PCR-based tissue screening panel indicates expression in humanleukocyte. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development. Antibody detection of circulating fragments of thefull-length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. If a disorder is characterized by a specific mutation in theprotein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in themalignant melanoma (metastatic to lymph node), brain (glioblastoma),thyroid, colon tumor (RER+), stomach (poorly differentiatedadenocarcinoma with signet ring cell features), primary B-cells fromtonsils, lung carcinoid, Burkitt lymphoma and human leukocyte. Thediagnostic uses can be applied, not only in genetic testing, but also inmonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the or relevant fragments can beused to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in the malignant melanoma(metastatic to lymph node), brain (glioblastoma), thyroid, colon tumor(RER+), stomach (poorly differentiated adenocarcinoma with signet ringcell features), primary B-cells from tonsils, lung carcinoid, Burkittlymphoma and human leukocyte. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the dehydrogenase to a binding partnersuch as a substrate. These uses can also be applied in a therapeuticcontext in which treatment involves inhibiting the protein's function.An antibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the peptides activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a dehydrogenase polypeptide of the present invention. Suchnucleic acid molecules will consist of, consist essentially of, orcomprise a nucleotide sequence that encodes one of the dehydrogenasepolypeptides of the present invention, an allelic variant thereof, or anortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB,particularly contiguous peptide encoding sequences and peptide encodingsequences within the same gene but separated by introns in the genomicsequence. The important point is that the nucleic acid is isolated fromremote and unimportant flanking sequences such that it can be subjectedto the specific manipulations described herein such as recombinantexpression, preparation of probes and primers, and other uses specificto the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. However, the nucleic acidmolecule can be fused to other coding or regulatory sequences and stillbe considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified partially or substantially) DNA molecules in solution. IsolatedRNA molecules include in vivo or in vitro RNA transcripts of theisolated DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule. The present invention further provides nucleic acidmolecules that consist essentially of the nucleotide sequence shown inFIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomicsequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consistsessentially of a nucleotide sequence when such a nucleotide sequence ispresent with only a few additional nucleic acid residues in the finalnucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

Full-length genes may be cloned from known sequence using any one of anumber of methods known in the art. For example, a method which employsXL-PCR (Perkin-Elmer, Foster City, Calif.) to amplify long pieces of DNAmay be used. Other methods for obtaining full-length sequences are wellknown in the art.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the dehydrogenase polypeptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding, and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form of DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention and that encodeobvious variants of the dehydrogenases of the present invention that aredescribed above. Such nucleic acid molecules may be naturally occurring,such as allelic variants (same locus), paralogs (different locus), andorthologs (different organism), or may be constructed by recombinant DNAmethods or by chemical synthesis. Such non-naturally occurring variantsmay be made by mutagenesis techniques, including those applied tonucleic acid molecules, cells, or whole organisms. Accordingly, asdiscussed above, the variants can contain nucleotide substitutions,deletions inversions, and/or insertions. Variation can occur in eitheror both the coding and non-coding regions. The variations can produceboth conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in the FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences, and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could be at least 30, 40, 50, 100250, or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encodeepitope-bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50, or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence.

FIG. 3 provides information on SNPs that have been found in the geneencoding the transporter protein of the present invention. SNPs wereidentified at 11 different nucleotide positions in introns and regions5′ and 3′ of the ORF. Such SNPs in introns and outside the ORF mayaffect control/regulatory elements. The changes in the amino acidsequence that these SNPs cause can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as abase.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 11 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asthose, which may encompass fragments disclosed prior to the presentinvention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.Moreover, the nucleic acid molecules are useful for constructingtransgenic animals wherein a homolog of the nucleic acid molecule hasbeen “knocked-out” of the animal's genome.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form, and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatdehydrogenases of the present invention are expressed in the malignantmelanoma (metastatic to lymph node), brain (glioblastoma), thyroid,colon tumor (RER+), stomach (poorly differentiated adenocarcinoma withsignet ring cell features), primary B-cells from tonsils, lungcarcinoid, Burkitt lymphoma detected by a virtual northern blot. Inaddition, PCR-based tissue screening panel indicates expression in humanleukocyte. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease indehydrogenase expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a dehydrogenase, such as by measuring alevel of a receptor-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a receptor gene hasbeen mutated. Experimental data as provided in FIG. 1 indicates thatdehydrogenases of the present invention are expressed in the malignantmelanoma (metastatic to lymph node), brain (glioblastoma), thyroid,colon tumor (RER+), stomach (poorly differentiated adenocarcinoma withsignet ring cell features), primary B-cells from tonsils, lungcarcinoid, Burkitt lymphoma detected by a virtual northern blot. Inaddition, PCR-based tissue screening panel indicates expression in humanleukocyte.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate dehydrogenase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe dehydrogenase gene, particularly biological and pathologicalprocesses that are mediated by the dehydrogenase in cells and tissuesthat express it. Experimental data as provided in FIG. 1 indicatesexpression in the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte. The method typically includes assaying the ability of thecompound to modulate the expression of the dehydrogenase nucleic acidand thus identifying a compound that can be used to treat a disordercharacterized by undesired dehydrogenase nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the dehydrogenase nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for dehydrogenase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the dehydrogenasesignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

Thus, modulators of dehydrogenase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of dehydrogenasemRNA in the presence of the candidate compound is compared to the levelof expression of dehydrogenase mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof nucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate dehydrogenase nucleic acid expression incells and tissues that express the dehydrogenase. Experimental data asprovided in FIG. 1 indicates that dehydrogenases of the presentinvention are expressed in the malignant melanoma (metastatic to lymphnode), brain (glioblastoma), thyroid, colon tumor (RER+), stomach(poorly differentiated adenocarcinoma with signet ring cell features),primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma detectedby a virtual northern blot. In addition, PCR-based tissue screeningpanel indicates expression in human leukocyte. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) of nucleic acid expression.

Alternatively, a modulator for dehydrogenase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thedehydrogenase nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data as provided in FIG. 1 indicatesexpression in the malignant melanoma (metastatic to lymph node), brain(glioblastoma), thyroid, colon tumor (RER+), stomach (poorlydifferentiated adenocarcinoma with signet ring cell features), primaryB-cells from tonsils, lung carcinoid, Burkitt lymphoma and humanleukocyte.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe dehydrogenase gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in dehydrogenase nucleic acid, and particularly inqualitative changes that lead to pathology. The nucleic acid moleculescan be used to detect mutations in dehydrogenase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the dehydrogenase gene and thereby to determine whether a subjectwith the mutation is at risk for a disorder caused by the mutation.Mutations include deletion, addition, or substitution of one or morenucleotides in the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns, or changes in gene copy number, such as amplification.Detection of a mutated form of the dehydrogenase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a dehydrogenase.

Individuals carrying mutations in the dehydrogenase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 11different nucleotide positions in introns and regions 5′ and 3′ of theORF. Such SNPs in introns and outside the ORF may affectcontrol/regulatory elements. The changes in the amino acid sequence thatthese SNPs cause can readily be determined using the universal geneticcode and the protein sequence provided in FIG. 2 as a base. Genomic DNAcan be analyzed directly or can be amplified by using PCR prior toanalysis. RNA or cDNA can be used in the same way. In some uses,detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a dehydrogenase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantdehydrogenase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., Biotechniques19:448 (1995)), including sequencing by mass spectrometry (see, e.g.,PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the dehydrogenase gene inan individual in order to select an appropriate compound or dosageregimen for treatment. FIG. 3 provides information on SNPs that havebeen found in the gene encoding the transporter protein of the presentinvention. SNPs were identified at 11 different nucleotide positions inintrons and regions 5′ and 3′ of the ORF. Such SNPs in introns andoutside the ORF may affect control/regulatory elements. The changes inthe amino acid sequence that these SNPs cause can readily be determinedusing the universal genetic code and the protein sequence provided inFIG. 2 as a base.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol dehydrogenase gene expression in cells, tissues, and organisms.A DNA antisense nucleic acid molecule is designed to be complementary toa region of the gene involved in transcription, preventing transcriptionand hence production of dehydrogenase. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into dehydrogenase.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of dehydrogenase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired dehydrogenase nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the dehydrogenase, suchas ligand binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in dehydrogenase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desireddehydrogenase to treat the individual.

The invention also encompasses kits for detecting the presence of adehydrogenase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that dehydrogenases of the presentinvention are expressed in the malignant melanoma (metastatic to lymphnode), brain (glioblastoma), thyroid, colon tumor (RER+), stomach(poorly differentiated adenocarcinoma with signet ring cell features),primary B-cells from tonsils, lung carcinoid, Burkitt lymphoma detectedby a virtual northern blot. In addition, PCR-based tissue screeningpanel indicates expression in human leukocyte. For example, the kit cancomprise reagents such as a labeled or labelable nucleic acid or agentcapable of detecting dehydrogenase nucleic acid in a biological sample;means for determining the amount of dehydrogenase nucleic acid in thesample; and means for comparing the amount of dehydrogenase nucleic acidin the sample with a standard. The compound or agent can be packaged ina suitable container. The kit can further comprise instructions forusing the kit to detect dehydrogenase mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides arrays or microarrays of nucleicacid molecules that are based on the sequence information provided inFIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet. al., U.S. Pat. No. 5,807,522.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides that are only 7-20 nucleotidesin length. The microarray may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull-length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene(s) of interest (or an ORF identified from thecontigs of the present invention) is typically examined using a computeralgorithm that starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide that preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large-scale correlation studies on the sequences,expression patterns, mutations, variants, or polymorphisms amongsamples.

Using such arrays, the present invention provides methods to identifythe expression of one or more of the proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention. FIG. 3 provides informationon SNPs that have been found in the gene encoding the transporterprotein of the present invention. SNPs were identified at 11 differentnucleotide positions in introns and regions 5′ and 3′ of the ORF. SuchSNPs in introns and outside the ORF may affect control/regulatoryelements. The changes in the amino acid sequence that these SNPs causecan readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a base.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid. Preferred kits will include chips that are capable of detectingthe expression of 10 or more, 100 or more, or 500 or more, 1000 or more,or all of the genes expressed in Human.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified dehydrogenase genes of the present invention canbe routinely identified using the sequence information disclosed hereincan be readily incorporated into one of the established kit formatswhich are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in procaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterodehydrogenase. Typical fusion expression vectorsinclude pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Ma.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Examples ofsuitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in a host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow etal., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufnan etal., EMBO J.6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance, propagation, or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced, or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing adehydrogenase polypeptide that can be further purified to producedesired amounts of dehydrogenase or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe dehydrogenase or dehydrogenase fragments. Thus, a recombinant hostcell expressing a native dehydrogenase is useful for assaying compoundsthat stimulate or inhibit dehydrogenase function.

Host cells are also usefil for identifying dehydrogenase mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutantdehydrogenase (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native dehydrogenase.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a dehydrogenaseand identifying and evaluating modulators of dehydrogenase activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the dehydrogenase nucleotidesequences can be introduced as a transgene into the genome of anon-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the dehydrogenase to particular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,dehydrogenase activation, and signal transduction, may not be evidentfrom in vitro cell-free or cell-based assays. Accordingly, it is usefulto provide non-human transgenic animals to assay in vivo dehydrogenasefunction, including ligand interaction, the effect of specific mutantdehydrogenases on dehydrogenase function and ligand interaction, and theeffect of chimeric dehydrogenases. It is also possible to assess theeffect of null mutations, which is mutations that substantially orcompletely eliminate one or more dehydrogenase functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention, which are obvious to those skilled inthe field of molecular biology or related fields, are intended to bewithin the scope of the following claims.

4 1 1335 DNA Human 1 tgcttccgga gccggagggg gcccggcgta cccagcccccagcccgacgt gaccatgctg 60 tcccgcctcc taaaagaaca ccaggccaag cagaatgaacgcaaggagct gcaggaaaag 120 aggaggcgag aggctatcac tgcagcgacc tgcctgacagaagctttggt ggatcacctc 180 aatgtgggtg tggcccaggc ctacatgaac cagagaaagctggaccatga ggtgaagacc 240 ctacaggtcc aggctgccca atttgccaag cagacaggccagtggatcgg aatggtggag 300 aacttcaacc aggcactcaa gctggaccag agaggcttccgagtcctggc cagctgcctg 360 accccctccg gggccgagga cctgcagcgg gtggcctcctcccgcctcca caccaccctg 420 ttggatatca ctgatcccca gagcgtccag caggcagccaagtgggtgga gatgcacgtt 480 aaggaagcag ggctttttgg tctggtgaat aatgctggtgtggctggtat catcggaccc 540 acaccatggc tgacccggga cgatttccag cgggtgctgaatgtgaacac aatgggtccc 600 atcggggtca cccttgccct gctgcctctg ctgcagcaagcccggggccg ggtgatcaac 660 atcaccagcg tcctgggtcg cctggcagcc aatggtgggggctactgtgt ctccaaattt 720 ggcctggagg ccttctctga cagcctgagg cgggatgtagctcattttgg gatacgagtc 780 tccatcgtgg agcctggctt cttccgaacc cctgtgaccaacctggagag tctggagaaa 840 accctgcagg cctgctgggc acggctgcct cctgccacacaggcccacta tgggggggcc 900 ttcctcacca agtacctgaa aatgcaacag cgcatcatgaacctgatctg tgacccggac 960 ctaaccaagg tgagccgatg cctggagcat gccctgactgctcgacaccc ccgaacccgc 1020 tacagcccag gttgggatgc caagctgctc tggctgcctgcctcctacct gccagccagc 1080 ctggtggatg ctgtgctcac ctgggtcctt cccaagcctgcccaagcagt ctactgaatc 1140 cagccttcca gcaagagatt gtttttcaag gacaaggactttgatttatt tctgccccca 1200 ccctggtact gcctggtgcc tgccacaaaa taagcactaacaaaagtgta ttgtttaaaa 1260 aataaaaaga aggtgggcag aaatgtgccc agtggaaaaaaaaaaaaaaa aaaaaaaaaa 1320 aaaaaaaaaa aaaaa 1335 2 360 PRT Human 2 MetLeu Ser Arg Leu Leu Lys Glu His Gln Ala Lys Gln Asn Glu Arg 1 5 10 15Lys Glu Leu Gln Glu Lys Arg Arg Arg Glu Ala Ile Thr Ala Ala Thr 20 25 30Cys Leu Thr Glu Ala Leu Val Asp His Leu Asn Val Gly Val Ala Gln 35 40 45Ala Tyr Met Asn Gln Arg Lys Leu Asp His Glu Val Lys Thr Leu Gln 50 55 60Val Gln Ala Ala Gln Phe Ala Lys Gln Thr Gly Gln Trp Ile Gly Met 65 70 7580 Val Glu Asn Phe Asn Gln Ala Leu Lys Leu Asp Gln Arg Gly Phe Arg 85 9095 Val Leu Ala Ser Cys Leu Thr Pro Ser Gly Ala Glu Asp Leu Gln Arg 100105 110 Val Ala Ser Ser Arg Leu His Thr Thr Leu Leu Asp Ile Thr Asp Pro115 120 125 Gln Ser Val Gln Gln Ala Ala Lys Trp Val Glu Met His Val LysGlu 130 135 140 Ala Gly Leu Phe Gly Leu Val Asn Asn Ala Gly Val Ala GlyIle Ile 145 150 155 160 Gly Pro Thr Pro Trp Leu Thr Arg Asp Asp Phe GlnArg Val Leu Asn 165 170 175 Val Asn Thr Met Gly Pro Ile Gly Val Thr LeuAla Leu Leu Pro Leu 180 185 190 Leu Gln Gln Ala Arg Gly Arg Val Ile AsnIle Thr Ser Val Leu Gly 195 200 205 Arg Leu Ala Ala Asn Gly Gly Gly TyrCys Val Ser Lys Phe Gly Leu 210 215 220 Glu Ala Phe Ser Asp Ser Leu ArgArg Asp Val Ala His Phe Gly Ile 225 230 235 240 Arg Val Ser Ile Val GluPro Gly Phe Phe Arg Thr Pro Val Thr Asn 245 250 255 Leu Glu Ser Leu GluLys Thr Leu Gln Ala Cys Trp Ala Arg Leu Pro 260 265 270 Pro Ala Thr GlnAla His Tyr Gly Gly Ala Phe Leu Thr Lys Tyr Leu 275 280 285 Lys Met GlnGln Arg Ile Met Asn Leu Ile Cys Asp Pro Asp Leu Thr 290 295 300 Lys ValSer Arg Cys Leu Glu His Ala Leu Thr Ala Arg His Pro Arg 305 310 315 320Thr Arg Tyr Ser Pro Gly Trp Asp Ala Lys Leu Leu Trp Leu Pro Ala 325 330335 Ser Tyr Leu Pro Ala Ser Leu Val Asp Ala Val Leu Thr Trp Val Leu 340345 350 Pro Lys Pro Ala Gln Ala Val Tyr 355 360 3 19806 DNA Humanmisc_feature (1)...(19806) n = A,T,C or G 3 nnnnnnnnnn nnnnnnnnnnnnnattctat ttaattagtc tggaatcaag gctttttttt 60 tttttttttt gagacagagtcccactctgt cgcccgggct ggagttcagt ggcgtgatct 120 cggctcactg caacctctgcctcccaggtt caagcgattc tcctgcctca ggctcctgag 180 tagctgggat tacaggcttgtgccactacg cctggctaaa ttttgtattt ttagtagata 240 caaggtttca ccatgttggtcagcctggtc tcgaactcct gacctcgtga tccgcccgcc 300 tcggcctccc aaagtgctgggattacgggc gtgagccacc gcacccggct aatcacggca 360 tttttataag ccttgtgatttctgtgattc taatgtttag attctaatgc ttagccaggg 420 ttgagaacca ccgatttaatccaatccctt ctcctagttt tactaaagag aaaactataa 480 cctagaatgg tgagcaacttgcccaaagtc acccaacatg ttagtgcaag gtgcaccgag 540 agagattgta ccagtagcaagaatatgctc atgatgttta taattgttct cgctggagtt 600 aatcccggaa gcatttctttttagttcaca gaggccttat ataaattact ttttactttg 660 gcacagcact tacgcttctgctaacactga aatgggttcg cattcctgac cacaaaagga 720 cagagatgaa attctacattcacacagccc gccaagttag ccaagctccc taggaggctg 780 tctgaagtgc ctaaaatgcttctctacaat gatcacccag agctgagaga cttcagtggg 840 gtagtgagaa gaaagagggttgggagagac aggaaagcat cctctccttg agggaaggaa 900 ctgggaatca actgagaaccagctagcact gccaggaggt gaggagaggg aaggagaata 960 atttaaatga ggccaggggagcttctgctc cctcaattaa acggtgatag acggcctgac 1020 accaccagcc ctcgaagcctgagatccaca ggaaatgtta aaaactggct tggcaatata 1080 agtattagaa aatacttcttccaacactca ccaaaaacta agctcccaat aaagaacact 1140 tcacctgccc tccgcaaccctctacctctc ttccccgcca agatcttcac ccaaggtctc 1200 aagagggcgg ttcccaacctcacgtgacac agcggtcacg tgacatggcc ccggggagcc 1260 gaggtgagcg ttccagcttccggagccgga gggggcccgg cgtacccagc ccccagcccg 1320 acgtgaccat gctgtcccgcctcctaaaag aacaccaggc caagcagaat gaacgcaagg 1380 agctgcaggg tgagccaaatatcctgtcgg ccgttttctc ttcggccgcg gcctagcttc 1440 agcccggagc ctggatctcgagtaactaac catatccagg gaaagacgcc agctagcggg 1500 caacgggcat gggggnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nntcccctcc aattcctttt ccccctctcc 3540 ccagcaaaat aggaggcgagaggctatcac tgcagcgacc tgcctgacag aagctttggt 3600 ggatcacctc aatgtggggtatggacctct tatcaacatc agtttcctcc ttccccaccc 3660 cgcccaagtt taggcactggccagtctggc cctcaaatag ctgttgaagg ggtgggatgt 3720 tccactaatt cccctatcctaccccgcccc tcccagctct ttgtagagca acttgagtca 3780 actctgagtc ctagcactgggcaagggagg aacagctgcc gtggttagag aagcagccag 3840 atttcccctt ccccacgttaacttccctgg catttacaac ttgatgccat ctgcccacct 3900 cccttcaccc ttccaagtccagctgtcact tcagcaggag ggagagcacc ctccttcatt 3960 acagcttacc accctctcctctgcctccca ccctctggca agcctgggga gcagctggca 4020 ggaaagagat ggcagagctggtggtggtga gagtagaacc tgttccggga gctatggcag 4080 agccaggctg tctcttaccttcctattggg tctctaggga ccacaccctg ccccagccct 4140 aaatgagaat gcaagtaacagccaaagact tgggaaaaag caaagaacat tgtctcttga 4200 ccctaagtga cccagaagcgtgcagagatg atgatttgct agtctgccta ttggaagaaa 4260 ggcagtatgg taccttccaccccaggtcaa gtagaacagc tcggtgtgaa tccagagact 4320 gagtcatcca agtgagccatgcaggggctg gggtcatctt tgttactcat cttgggggaa 4380 ggttgagaga aagaaagttgtggctggggc ctctgatctc ccttctctcc aggcagctct 4440 ctttactcag tgtgaatatagaagcaggtg gtcaatgggg aaaaccagaa gttcaggaat 4500 tctcagggga gtctgtttcagttcctaccc gacccttgac agtgacccag ctgtctccca 4560 aaaagaagga acagggtctgccctcccatt tcctccctcc cacattggca cctcctgggc 4620 tctgctgtgc ccatcatttgtgagattggc ccaggccttt ccctcttctt ttcctttgct 4680 agatgccacc ccactttcagcttagagggc agctaagcca aagccagatt agaaagggtt 4740 ttgtgttgct gcccacgcctcctctcattc cccggaaagg aaaacaaagg ctcagtctat 4800 cttggcccct gtcaggtgtcctgcccactc cctcagcccc caccaacccc ttccccgctc 4860 cagcccccac acattccagtgggtgggggc accggatgtg gaatctcctg gctgagtaga 4920 gctctggggt gggaagtgaaaaattcaaca gccaataaag gagaacaatt attgcagggg 4980 ttggggaggg caaaaaacactggcagaagg ttggggacac caaccccatg gtagtaatgg 5040 taaccacagc ccataccttgattgaaaaga aaaactagtg cctaaggcag aagggaggga 5100 gagcatgtgt gtgtgtgtgtgtgtgtgttt gtgtgtgttc cttgatctgt gtgggcaaaa 5160 gcgaaggctt gggagagcaactgagagccg agaagaaacc cctgggatac cctcttttga 5220 cccagggttc ctggggagggggtttgtact cccatcctaa cccggcttca gggaggggcc 5280 caatttccct ctccaacttcttgcatagat ccctaggctt ccaatcactg ccagatgtgt 5340 tcctcctgct ggnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc 5460 caggttttcc ctcccttcccccactcagct gcaggaactc ctttttgggg tttggagctg 5520 gtatgtttct agtcagctccgagcttggct ctcctgggaa tcctgggagt gaaaggaagg 5580 agctgggttt atttgcatgtactggtagtc atttgcatca catccaaaaa tggccaaaat 5640 tatgagccct gattcttggctgaactccca ctgctgcaat ggaatattag tcccggagac 5700 cacccccaac tagctggagctgatctcctc cctcctccaa ccccccagtg tggcccaggc 5760 ctacatgaac cagagaaagctggaccatga ggtgaagacc ctacaggtcc aggctgccca 5820 atttgccaag cagacaggccagtggatcgg aatggtggag aacttcaacc aggcactcaa 5880 ggtgggccat actccctacctcaccacccc aatcctgggc ccccattggc tgcctccagt 5940 caggttacct caggtttaggttaaggagga agtagggtgg tcccagaaac cccatctata 6000 gccccagtgt cagaaaaggtagagaaagaa agaaaagcag ttggtgggtc caagtaaagc 6060 cttttccagg agatgaataaaacgtattcc ccagactgga agccatactc tacccattct 6120 gattcctggg ctcccacctcctctccccct tcccaggaaa ttggggatgt ggagaactgg 6180 gctcggagca tcgagctggacatgcgcacc attgccactg cactggaata tgtctacaaa 6240 gggcagctgc agtctgccccttcctagccc ctgttccctc ccccaaccct atccctccta 6300 cctcacccgc agggggaaggagggaggctg acaagccttg aataaaacac aagcctccgt 6360 ttctctgtgg tgtgtttcagagagctacta gctccagtgt cgggggtggg agtggaaggt 6420 tcaaaggtgg tttccctgagggacaggtac cttttgggga gagggtggaa ctagcttcct 6480 cttactatcc caactctcttctcctccatg gcccttgtgc aggtgtctgt taggcaagca 6540 gagggtggga gttcccatccctcctgagag aaggtcctag tagccctgcc ccaagcttcc 6600 taattcagga cttgtttcctacagaagaga aacaaggcaa ggtacaggcc tggtccccag 6660 ctctggcttt ctgcctctccacgtgctcat ggcctctccc aggctaactc taagcagtgt 6720 catgagtctg agccaggtgggagattaatt cctgggggca cttcagggct gagaaggggg 6780 aggaatgaca ggtccagtaaccgttaccaa cagagcagtg cagctgccat ccttgacagc 6840 tccctcctcc ttggagaccatgacatagat ggtcaggaac ccaggctgag aaagacagcc 6900 aaggggtggg gggagcctaggcaaatctgg cctctgccaa gtcctggctt cagccaggca 6960 agctccagcc tccctggctcctcctcctcc tcagtcctat ccccaccctg tcacacatac 7020 acttaatacg cctggcatccaagtccaccc actccggact ttggccttag cagtagttag 7080 tgtgggaggc tgggaagactgggagcagtc tcttaaacaa aagcaaaaga ataagcttcg 7140 ggcgctgtag tacctgccagctttcgccac aggaggtaag tggatactgg gagctggggg 7200 aactgagaag actagccagatattacatgt attgccaact caaaactttc agcttttaac 7260 atgcttcctc acacattatcccctttgatc ctccacaact ctgaggtgga cctggtgggt 7320 cttagcccca cttggtagatgagaaaatag gttgagagag acagtgagat gctcagtatc 7380 acacagcaaa cctcttggccctatacatca ttccaaacac aagacccagg ttgcatatag 7440 aaggttcagt gtccctggtttagaaggaga ggtggtgtga ggcaagcaag aagatgcctc 7500 tgctgcactc cagcctgggcgacagagtga gactccatct caaaaaaaaa aaaaaaaaaa 7560 aaaagatgcc tctgctccatacagcaggtc tgtacacagg atctggctca tgtggtttta 7620 gttnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7680 nnnnnnnnnn nnnnnnnnnntggttttagt taaagttagc cacaaataca gcgtctgccc 7740 acatctttgc tttgaacagatgagccatgg ttggccaatt atctgccaac cagataattt 7800 ctcaatatgc tcacaccagatgcttccagc tagggagggt attaggggaa agggcttgag 7860 ggccacagta aactggacaagtttttctgc ccagcctagg ctgccacctg taggtcactt 7920 gggctccagc tatgtggctgcctcttctgc tgggtgcctt actctgggca gtgctgtggt 7980 tgctcaggga ccggcagagcctgcccgcca gcaatgcctt tgtcttcatc accggctgtg 8040 actcaggctt tgggcgccttctggcactgc agctggacca gagaggcttc cgagtcctgg 8100 ccagctgcct gaccccctccggggccgagg acctgcagcg ggtggcctcc tcccgcctcc 8160 acaccaccct gttggatatcactgatcccc agagcgtcca gcaggcagcc aagtgggtgg 8220 agatgcacgt taaggaagcaggtaagtatg gtagaccacc aggaatatgg tgtggggtgt 8280 cctgatcccc acagtcaccccaggagtcac ctgcaagggc tgtggtaagc taaagggaca 8340 atttgaggag aagcagttttcagatgctcc caggaagaag agggagctgt gggagtgcct 8400 cacctacccc cagcatccttttcatctccc cacagggctt tttggtctgg tgaataatgc 8460 tggtgtggct ggtatcatcggacccacacc atggctgacc cgggacgatt tccagcgggt 8520 gctgaatgtg aacacaatgggtcccattgg ggtcaccctt gccctgctgc ctctgctgca 8580 gcaagcccgg ggccgggtgatcaacatcac cagcgtcctg ggtcgcctgg cagccaatgg 8640 tgggggctac tgtgtctccaaatttggcct ggaggccttc tctgacagcc tgaggtgagg 8700 ggtacagggc tctgggttccaggactaaca gcagcccact caacaaacgt gggccagcag 8760 aggtggttaa aatacagcacattggaatag ttaaaaagag acagtttagg gctaaacttc 8820 atgggttcaa tgaagtctacccttatgtaa gctttgtgac nnnnnnnnnn nnnnnnnnnn 8880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn ttcttagcag gagtataagg cgcctaagct 16020 tagtctacct tttaaggaagcctgcgtttg tcaccatcac tcagcaaata acctgaatgt 16080 ctcctgtctc tcagccttaatttttcaggc agcatcatgg gacacatact tttagttttg 16140 agacaaggcc ttgctctcacccagggtgga gtgcagtggt gcagtcacgg cccactgaac 16200 ttcaaactcc taggctcaagcagctcaagc gatatccgcc tcagcctcct gagtagctga 16260 gaccacaggc gcgtgccagcatgcctggct agtatttttt tacagatggg gtcttgctgt 16320 ggtgaccaga cttgtctcaaactcccggcc tcaagcgatg cttccgcctg ggcctcccaa 16380 agtgttggga ttataggtgtgagccactgc atactggaac acatacttta tacttgaatt 16440 tttttttatc cccttccctcctgctcctta cctatacttg gatttctaca tctgtgccag 16500 ggcagtggga tgtatccccactttccccat cagcttaccc tccagcaaat acgagactat 16560 acccttcaat atccagcactcagggctcaa ccatgtgttt tgggagcaag ggaatggggt 16620 tcctctaggt caggaatcggcaaactcagt actcaagcca gatctggcca gctgcctaca 16680 agctgataat ggttttttttatttttaaat ggttacattg taaactgtta tataagtacc 16740 tgataatatc attaattttgtttcttggcc tgccatgctt aaaatattaa ctctctggcc 16800 ctttaagaaa aaaacgtgctgacccctgct ctagatcaaa gaaaacaaac ctcaaaaata 16860 ctttcctccc tctaccccacttgacccttg tcccggggca gtaggcatct ccgtcaaaac 16920 tcttgtccct ggtctgtggtaactttctca gctccccaac ccatgtccct caaagtcccc 16980 tccctatagg gcaagaacccagcaacttcg ctctgccccg actctaggcg ggatgtagct 17040 cattttggga tacgagtctccatcgtggag cctggcttct tccgaacccc tgtgaccaac 17100 ctggagagtc tggagaaaaccctgcaggcc tgctgggcac ggctgcctcc tgccacacag 17160 gcccactatg ggggggccttcctcaccaag tgtgagtagc caggcccaca caggggcaca 17220 tgaagggaaa caagtaccagaaaggccagt cctgcataag cctgctagga ggtgggtggg 17280 gcacccaggg cagggttgagggtgaacagg atgttacaan agtgcccagg ccatgtggaa 17340 cctccccact ccccacactgaggaggggac tgagggtgac aagcccaggg ccccagaaga 17400 cagtacctaa gatgggctggagtgaggaag ggaaactgat tgcaaccacc tatggggctg 17460 cagacctgaa aatgcaacagcgcatcatga acctgatctg tgacccggac ctaaccaagg 17520 tgagccgatg cctggagcatgccctgactg ctcgacaccc ccgaacccgc tacagcccag 17580 gttgggatgc caagctgctctggctgcctg cctcctacct gccagccagc ctggtggatg 17640 ctgtgctcac ctgggtccttcccaagcctg cccaagcagt ctactgaatc cagccttcca 17700 gcaagagatt gtttttcaaggacaaggact ttgatttatt tctgccccca ccctggtact 17760 gcctggtgcc tgccacaaaataagcactaa caaaagtgta ttgtttaaaa aataaaaaga 17820 aggtgggcag aaatgtgcccagtggaaggc tgaccccatt taagtgccaa ctactccaaa 17880 ccgacatgct cacggtctctggcctgttca gtccctgcaa aacagctagc acccacagtg 17940 gggcgccagg gaactgcctcacatctacag ctgcacgtcg gggagtggcc atcaaagggc 18000 actttaatac atttcccttattttctgaag gggagtaagg ttgcaattca gtgtctgtac 18060 tgggaatggt cttcatatttcttgggggag aagagcaggt gatgagggtt ctgggccagg 18120 ctgggtggct tccatggaagaaaaggcaat attcacataa attctcctgc taaggacact 18180 gaccacacag gtgtcaaggcaacttatcat acttcgaaag gagctggatc ccttgaggat 18240 tggccaggaa gggaggtgctgggcccttag cggtgcacag aaggccagga agatgtccaa 18300 ggcagatggg ggctgggctctcgcaggtgg gacctttctg gggagctgct ttgacttatg 18360 cagcagatgg cttcatgaatgttcatagtg agcctggcag cataagacta gggggcagaa 18420 agcaccacag tctctggatcctcacttctc ccactgcctg cccaaccaac accttcgcaa 18480 agtcctcctt tcccaaacaccccccaaaat agacctcgaa gtacacatgc attaaggtcc 18540 cagaggacag ggaacatcagtaaggaaagg aaggaatcaa gcatcactct aagacaaact 18600 cagaccatct cttttcggtctgaaaaaata atccgtttaa ttgaaaaacc tggaggatac 18660 tattccactc ccccagatgaggaggctgag gagaccagac ccctacatca cctcgtagcc 18720 acttctgata ctcttcacgaggcagcaggc aaagacaatt cccaaaacct agaaggaaag 18780 atggggacag gggtggagaggagtcagaag ggctagctac ctcagaccca tgcaagagac 18840 tccaaacaca cactcccaggccagtacctc ctgttcagca ccctcctccc ctcagccccc 18900 ttccctccag gcaaccctggacagagtccc agcccctgct cagggttatc tcttacctcg 18960 acaaaagcaa ttccaagggctgctgcagct accaccagca catttttcct cagccagccc 19020 ccaatcttct ccacacagccctgaaggtgg caggcacaaa ggacaaaagc accatcagaa 19080 acttcccacc ccaaccccctcccttggtcc atcagtctct tccctgcccc tgtacctttc 19140 accccaccct tagcatttccagatcccctc cccatccctg accctgtcca cctccatcct 19200 gggtctcttg cattctaaaacatttcccag gtttcccaag tcccatacac agtctcctcc 19260 ctacctcctt atggatcgccttctcgttga aattaatccc acagcccaca gtaacattaa 19320 tgcagcagga gtcggggactcggttcttcg acatggaagg gattttctcc caatctgtgt 19380 agttagcagc cccacagcacttaaactggg ggagggaaga aatatggaga agaaggttgt 19440 taagtgaacc ttcaccttcaatatggagat gagaattctt tttttttttt tttttttttt 19500 tttgagacag agacttgctctgttgcccag acaagtgccc agcggtggct caatctcggc 19560 tcactgcaac ctccgcctcccgggttcaag cgattctcct gcctcaccct cctgagtagg 19620 tgggattaca ggcactcaccaccacacctg gctaattttt gtatttttag tagagacagg 19680 gtttcaccac gttggccaggctagtcttga acttctgacc tcaggtgatc tgcccacctc 19740 ggccttcaaa agtgctgagattacaggcgt gagtcaccac acccagcctt ggagatgaga 19800 attctc 19806 4 274 PRTHuman 4 Ala Leu Gln Leu Asp Gln Arg Gly Phe Arg Val Leu Ala Ser Cys Leu1 5 10 15 Thr Pro Ser Gly Ala Glu Asp Leu Gln Arg Val Ala Ser Ser ArgLeu 20 25 30 His Thr Thr Leu Leu Asp Ile Thr Asp Pro Gln Ser Val Gln GlnAla 35 40 45 Ala Lys Trp Val Glu Met His Val Lys Glu Ala Gly Leu Phe GlyLeu 50 55 60 Val Asn Asn Ala Gly Val Ala Gly Ile Ile Gly Pro Thr Pro TrpLeu 65 70 75 80 Thr Arg Asp Asp Phe Gln Arg Val Leu Asn Val Asn Thr MetGly Pro 85 90 95 Ile Gly Val Thr Leu Ala Leu Leu Pro Leu Leu Gln Gln AlaArg Gly 100 105 110 Arg Val Ile Asn Ile Thr Ser Val Leu Gly Arg Leu AlaAla Asn Gly 115 120 125 Gly Gly Tyr Cys Val Ser Lys Phe Gly Leu Glu AlaPhe Ser Asp Ser 130 135 140 Leu Arg Arg Asp Val Ala His Phe Gly Ile ArgVal Ser Ile Val Glu 145 150 155 160 Pro Gly Phe Phe Arg Thr Pro Val ThrAsn Leu Glu Ser Leu Glu Lys 165 170 175 Thr Leu Gln Ala Cys Trp Ala ArgLeu Pro Pro Ala Thr Gln Ala His 180 185 190 Tyr Gly Gly Ala Phe Leu ThrLys Tyr Leu Lys Met Gln Gln Arg Ile 195 200 205 Met Asn Leu Ile Cys AspPro Asp Leu Thr Lys Val Ser Arg Cys Leu 210 215 220 Glu His Ala Leu ThrAla Arg His Pro Arg Thr Arg Tyr Ser Pro Gly 225 230 235 240 Trp Asp AlaLys Leu Leu Trp Leu Pro Ala Ser Tyr Leu Pro Ala Ser 245 250 255 Leu ValAsp Ala Val Leu Thr Trp Val Leu Pro Lys Pro Ala Gln Ala 260 265 270 ValTyr

That which is claimed is:
 1. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes a protein comprising theamino acid sequence of SEQ ID NO:2; (b) a nucleic acid moleculeconsisting of the nucleic acid sequence of SEQ ID No: 1; (c) a nucleicacid molecule consisting of the nucleic acid sequence of SEQ ID No: 3;and (d) a nucleotide sequence that is completely complementary to anucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising anucleic acid molecule of claim
 1. 3. a vector according to claim 2,wherein said vector is selected from the group consisting of a plasmid,virus, and bacteriophage.
 4. A vector according to claim 2, wherein saidisolated nucleic acid molecule is inserted into said vector in properorientation and correct reading frame such that the protein of SEQ IDNO: 2 may be expressed by a cell transformed with said vector.
 5. Avector according to claim 4, wherein said isolated nucleic acid moleculeis operatively linked to a promoter sequence.
 6. A host cell containingthe vector of claim
 2. 7. A method for detecting the presence of anucleic acid molecule of claim 1 in a sample, said method comprisingcontacting the sample with an oligonucleotide comprising at least 20contiguous nucleotides that hybridizes to said nucleic acid moleculeunder stringent conditions, wherein the stringent condition ishybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SCC, 0.1% SDS at 50-65° C.,and determining whether the oligonucleotide binds to said nucleic acidmolecule in the sample.
 8. A process for producing a polypeptidecomprising culturing the host cell of claim 6 under conditionssufficient for the production of said polypeptide, and recovering thepeptide from the host cell culture.
 9. An isolated polynucleotideconsisting of a nucleotide sequence set forth in SEQ ID NO:1.
 10. Anisolated polynucleotide consisting of a nucleotide sequence set forth inSEQ ID NO:3.